This Phase II proposal will complete the development of an innovative sequencing platform (NPSeq) based on feasibility demonstrated in Phase I. This platform uses a nuclease protection assay combined with universal PCR to produce targeted sequencing libraries. The lysis-only protocol means there is no need to extract RNA or DNA or to reverse transcribe RNA. Consequently, sequencing from fixed tissue is as sensitive and accurate as sequencing unfixed tissue, addressing an unmet need for drug discovery, translational research, and diagnostics and making available vast amounts of archived clinical fixed tissue for which patient outcomes are known but are difficult to sequence via RNAseq. Comparative performance of NPSeq using FFPE vs. matched unfixed tissue results were better than RNAseq measurements from RNA extracted from the same samples. NPSeq is quantitative, sensitive, and highly reproducible with CV's of 3%, whether 10 or up to a thousand or more genes are measured. As a result, the same assay/platform can be used for biomarker identification as well as for verification, validation, and the final focused application measuring just a few genes, whether it is a drug discovery assay or an in vitro diagnostic (IVD). We demonstrated this capability with an assay of the whole miRnome of 1,942 miRNA from miRbase 18, overachieving on the Phase I Aims. We also demonstrated that mRNA and miRNA can be measured in the same assay, which is important: while miRNA can be measured from FFPE with greater success than mRNA, housekeeper mRNA genes need to be measured because the amount of diseased, non-necrotic tissue in each sample can be highly variable. Thus, the capability for dual measure of mRNA/miRNA can improve data and exploit the intrinsic (3%) reproducibility of the method. In this Phase II we will optimize, verify and validate the mRNA and miRNA commercial protocols, and develop protocols to measure mRNA mutations that are drug targets, such as gene fusions and expressed SNPs (eSNPs). We will generate an updated, whole-miRNome NPSeq assay (the miRbase19+ assay). We will also identify usable probes for the entire transcriptome and establish a database of gene-disease associations; these two tools together will allow for rapid development of customizable assays. These tools will be used to generate several assays during Phase II. We will then go through product development with the miRbase 19+ and several of the mRNA assays - generating data with cell lines and matched frozen and fixed clinical tissue to demonstrate the utility of these NPSeq assays and proceeding to kit development and product release. We expect that assays will provide investigators a pipeline for discovery, verification, validation, and application of biomarkers for their research or as diagnostic assays.